CasestudyonassemblingandassessingAOPinformation:AlkylationofDNAleadingtoheritablegeneticeffects

CaroleL.YaukEnvironmentalHealthScienceandResearchBureau

HealthCanadaOttawa,ON

Carole.Yauk@canada.ca

Outline

•  Review-MIE,KE,AO,KERsandtheAOP•  IntroductionandbackgroundrelevanttocasestudyAOP•  Selectionandsummaryofevents•  Summaryofweightofevidenceforkeyeventrelationships

•  Biologicalplausibility,empiricalsupport,uncertainties/inconsistencies

•  Assessmentofoverallweightofevidence•  Biologicalplausibility,empiricalsupport,essentiality

•  Conclusions

HepatocyteReducedVTGproduction

OvaryImpaired

OocyteDev.

FemaleDecreased

ovulation/spawn

PopulationDecliningTrajectory

AromataseInhibition

GranulosaReducedE2synthesis

PlasmaReduced

circulatingE2

WhatareAOPs?

• Awaytoorganizeinformation• Basedonbiologicalplausibilityand/orstatisticalinference• Ahypothesisandasetofmeasurementswecanmaketotestthathypothesis

• Eventsspanlevelsofbiologicalorganisationrelevanttoriskassessment(e.g.,molecular,cellular,tissue/organ,organismand/orpopulation).

AnAdverseOutcomePathway(AOP)isaconceptualframeworkthatportraysexistingknowledge

concerningthelinkagebetweenadirectmolecularinitiatingeventandanadverseoutcome,ata

levelofbiologicalorganizationrelevanttoriskassessment(Ankleyetal.2010,Environ.Toxicol.

Chem.,29(3):730-741.)

FivePrinciplesofAOPDevelopment

1. AOPsarenotchemicalspecific

2. AOPsaremodular

3. IndividualAOPsareapragmaticsimplificationofbiology

4. AOPnetworksarethefunctionalunitofprediction(inmostcases)

5. AOPsare“living”documents

PrinciplesofAOPDevelopment

KeyEvents(KEs)

• Functionalunitofobservation/verification• Observable∆biologicalstate(measurable)• Essential(butnotnecessarilysufficient)

TwoPrimaryBuildingBlocks

AOMIE

KeyEventsRelationships

(KERs)• Functionalunitofinference/extrapolation• directedrelationship• StateofKEupprovidessomeabilitytopredictorinferstateofKEdown

• Supportedbyplausibilityandevidence• Quantitativeunderstanding

Section6.

KERPages• Title• Description• Biologicalplausibility• Empiricalsupport• Inconsistenciesanduncertainties

• Quantitativeunderstanding

Plausibility• largelybasedon“normalbiology”• UnderstandingofstructuralandfunctionalrelationshipsbetweenKEs.

EmpiricalSupport• Largelybasedonperturbationstudies(e.g.,toxicologystudies)

WOEAssembly

CreatingKERs

OverallassessmentofAOPsWeightofevidenceassessmentforcausality

BradfordHillcriteria:minimalconditionsnecessarytoprovideevidenceofacausalrelationshipbetweeneventsandtheconsequenceformingtheweightofevidenceconsiderationsforcausality&levelofconfidenceinanAOP.

ER

Antagonism

HepatocyteReducedVTGproduction

OvaryImpaired

OocyteDev.

FemaleDecreasedovulation/

spawning

PopulationDecliningTrajectory

Aromatase

Inhibition

GranulosaReducedE2synthesis

AR

Agonism

Hypothalamic Neurons

(-)Feedback

CYP17,CYP11A

Inhibition

PlasmaReduced

circulatingE2

Reducedbonedensity

Osteoperosis

CorporaAllataReducedJHsynthesis

HemolymphReducedJH

concentrations

FatBodyReducedVTGproduction

Poornutritionalstatus

Formostreal-worldapplications,AOPnetworksarethefunctionalunitofprediction

4.EnteryourAOP,bylinkingtoexistingcontentand/orcreatingnewpagesasappropriate.

3.IdentifyexistingcontentyourAOPcanlinkto.

2.Search/browsethewikiforrelatedAOPs/KEs/KERsbeforeenteringnewAOP.

1.MapoutyourAOPaheadoftime

RecommendedWorkflowforAOPCreation

IMPORTANT BECAUSE: Germline mutations are deleterious and can result in death of the developing embryo. May result in any type of genetic disease in an offspring. Can potentially contribute to the population gene pool.

CASESTUDY:AlkylationofDNAleadingtoheritablegeneticeffects

Principle:Germlinegeneticdamageoccursinspermoreggandcanbetransmittedtoanoffspring

AOP:AlkylationofDNAleadingtoheritablemutations

Motivation:•  Oneofthebestcharacterizedmodesofactioningenetic

toxicology.•  Providecontextofusefornewmethods/technologiestodetect

somaticandgermcellmutations.•  Emphasizegapsinresearchinthisfield.•  Denovomutationsareincreasinglyrecognizedascontributing

toalargearrayofhumangeneticdiseases.Proposedchangesinthewayheritablehazardsareassessedarebeingconsidered.

BackgroundonbiologyofthisAOP

•  Alkylatingagents:chemicalsthataddalkylgroups(e.g.,methyl,ethyl,propyl)tocellularmolecules.

•  OccursatvarioussitesinDNA.Eachsitehasadifferentdegreeofstability.

© 2013 Nay SL, O'Connor TR. Published in [short citation] under CC BY 3.0 license. Available from: http://dx.doi.org/10.5772/54449

Prototypealkylatingagent

•  AlthoughAOPsarenotchemical-specific,thedatabaseonalkylationofDNAisheavilybiasedtowardsafewprototypeagents.

•  E.g.,N-ethyl-N-nitrosourea(ENU)

Alkyladductscanberepaired

•  PrimaryrepairproteinisAGT:Alkylguaninetransferase.•  AGTirreversiblybindsthealkylgroupandisinactivated.•  VeryefficientrepairatLOWdoses.•  AGToverwhelmedatHIGHdoses=alkyladductsretained.•  Notverygoodatrepairingalladducttypes.

ReplicationofalkylatedDNAcausesmutations

•  ReplicationoveranalkyladductcancauseinsertionofanincorrectbaseintheDNAduplex.

• Mutationbecomes‘fixed’andcanpropagatetodaughtercells.

O4thyminealkylation=AT-GCtransitionsO6guaninealkylation=GC-ATtransitions

Note:someadductsnotmutagenic.N-alkyladductstendtobebypassederror-free

TARGETCELLS:Spermatogonialstemcells

•  Mutationsinpre-meiotic,replicating,spermatogonialstemcellscanpersistinanorganismandclonallyexpand.

•  Spermderivedfromthesestemcellswillcarrythesemutations.•  Fertilizationofaneggwithspermcarryingmutationscanresultinanoffspringwiththesemutations.

Selectionofmeasurableendpoints

* Adduct✔

* Notrepaired✔

Repaired✔

Mutations(somatic)✔Mutations(sperm)✔

Replication✗Spermwithmutation✗ fertilizingegg

Offspringwithmutations✔

MolecularInitiatingEvent

CellularResponse

OrganismResponse

AlkylationofDNA

(Malepre-meioticgerm

cells)

Offspring:Increasednumbersof

mutationsinalltissues

Mutations

InsufficientorincorrectDNArepair

Inheritedmutations

KER1 KER2 KER3

KER4KER5

MIE AOKE1 KE2

AOP:AlkylationofDNAleadingtoheritablemutations

EntryofinformationintoOECDknowledgebase

KEs

KERs

Networkview

EntryofinformationintoOECDknowledgebase

WeightofEvidenceforKeyEventRelationships

1.   Biologicalplausibility•  Eacheventiswellcharacterizedandunderstood.Highlybiologicallyplausible.

2.   Empiricalevidenceinsupportofrelationships•  FindevidencetosupportthatKE1occursbeforeKE2.•  FindevidencetosupportthatincidenceKE1>KE2.•  Examineconcordanceofdose-andresponse–responserelationships

KeyEventRelationshipsEvaluation

AlkylationofDNA

Offspring:Increasednumbersofmutationsinalltissues

Mutations

Inheritedmutations

KER4KER5

MIE AOKE2

Temporalconcordance

Adducts:Peakearly,somepersistto6d

Mutationsinsperm:Exposed28d,mutationsoccur>49dayspost-exposure

Seileretal.1997.Mutationresearch385(3):205-211.

DataderivedfromO’Brienetal.2015EnvironMol.Mutagen.56(4):347-55.

Incidenceconcordance

Adducts>Mutations

Swenbergetal.(2008)ChemResToxicol21(1):253-265.

Incidenceconcordance:consolidatedENUdata

Adducts>>

Mutationsinsperm≥

Mutationsinoffspring

DataderivedfromYauketal.EnvironMolMutagen.2015.doi:10.1002/em.21954.

Incidenceconcordance:graphical

DataderivedfromdatainYauketal.EnvironMolMutagen.2015.doi:10.1002/em.21954.

Consideruncertaintiesandinconsistencies

•  Datagaps–empiricalevidencecomesalmostentirelyfromthechemicalENUingermcells.

•  Someinconsistenciesacrossexperimentsduetosub-standardprotocols.

•  Someinconsistenciesinincidenceconcordance–notappropriatetoquantitativelycomparesomeendpoints.

•  Differencesacrossexperimentsinchemicaldeliverymadecomparisonsdifficult.

Assessmentofoverallweightofevidence

•  AOPbasedon32highqualitystudiesacrossthreerodentspecies,fliesandfish.

•  Nosinglestudycomparedmultipleevents,butextrapolationscouldbemadeacrossstudies.

Assessmentofoverallweightofevidence

BiologicalPlausibility:IsthereamechanisticrelationshipbetweenKEupandKEdownconsistentwithestablishedbiologicalknowledge?”• Broadunderstandingandextensiveknowledgeforallofthekeyeventrelationships.• Generallywidelyaccepted.• Strong.

AssessmentofoverallweightofevidenceEssentiality:AredownstreamKEsand/ortheAOpreventedifanupstreamKEisblocked?•  EvidencesupportthatovercomingDNArepairisrequiredfor

mutationtooccur.•  Knock-downDNArepair,mutationsincrease;over-expressDNA

repair,mutationsdecrease.•  Moderate.

Figurederivedfromdatain:Allayetal.(1999)Oncogene:18(25):3783-3787.

AssessmentofoverallweightofevidenceEmpiricalsupport:DoesempiricalevidencesupportthatachangeinKEupleadstoanappropriatechangeinKEdown?DoesKEupoccuratlowerdosesandearliertimepointsthanKEdownandistheincidenceofKEup>thanthatforKedown?• Strongprimarilyfortheindirect(non-adjacent)KERs• UseofOECDtestguidelineor‘goldstandard’approachesindatacollection.• Highqualitystudies.• Overall:moderate–strong.

Conclusions

•  CurrentlyleveragingKEsandKERstobuildotherAOPs.

•  https://aopkb.org/aopwiki/index.php/Aop:15•  Seealso:Yauketal.Developmentoftheadverseoutcomepathway

"alkylationofDNAinmalepremeioticgermcellsleadingtoheritablemutations"usingtheOECD'susers'handbooksupplement.EnvironMolMutagen.56(9):724-50. 2015ANDYauketal.,AdverseOutcomePathwayonAlkylationofDNAinMalePre-MeioticGermCellsLeadingtoHeritableMutations.OECDSeriesonAdverseOutcomePathwaysISSN:2415-170X.http://dx.doi.org/10.1787/2415170X